Melanoma antigen peptide and uses thereof

ABSTRACT

The present invention relates to a melanoma antigen peptide comprising the amino acids sequence selected in the group consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5 or a function-conservative variant thereof. Moreover the invention also relates to a melanoma antigen peptide according to the invention for use in the prevention or the treatment of melanoma in patient.

FIELD OF THE INVENTION

The present invention relates to a melanoma antigen peptide comprisingthe amino acids sequence selected in the group consisting of SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5 or afunction-conservative variant thereof. Moreover the invention alsorelates to a melanoma antigen peptide according to the invention for usein the prevention or the treatment of melanoma in patient.

BACKGROUND OF THE INVENTION

In antitumor immune responses, CD8 cytotoxic T lymphocytes (CTL) havebeen identified as the most powerful effector cells (Vesely M D et al.,2011). As a consequence, most previous anti-cancer vaccines use class IHLA-restricted peptides derived from tumor antigens in order tostimulate CTL responses. However, the clinical impact of peptide-basedcancer vaccines remains still modest, even if a recent gp100-derivedpeptide vaccination was shown to increase patient survival in melanoma(Rosenberg S A et al., 2004 and Schwartzentruber D J et al., 2011). Inaddition to a variety of immune suppressive mechanisms originating fromthe tumor itself, suboptimal design of vaccines used so far may explainthis failure. In particular, short epitopic peptides, could inducevanishing CTL responses or tolerance towards targeted antigens (Bijker MS et al., 2007 and Toes R E et al., 1996). In the meanwhile, CD4 helperT cells have gained interest in anti-tumor immunity and immunotherapy.Indeed, tumor-reactive CD4+T helper 1 T cells (Th1) produce severalcytokines (such as IFN-γ, TNF-α and IL-2) essential for the induction ofcell-mediated immunity against tumors. One widely accepted modeldemonstrates the ability of CD4+ T cells to ‘license’ dendritic cells(DCs) for efficient CD8+ T cell priming through the interaction ofcostimulatory receptors (Bennett S R et al., 1998 and Smith C M et al.,2004). The cytokines secreted by CD4+Th1 cells also exert directantitumor and antiangiogenic effects. Furthermore, it has beendemonstrated in a mouse model that only tumor-reactive CD4+ T cells havebeen found to ensure efficient effector CTLs recruitment at the tumorsite. In a clinical standpoint, a high density of tumor-infiltratingCD4+ Th1 cells has been recently shown as a good prognostic marker incolorectal cancer patients emphasizing the role of these cells in cancerimmunosurveillance. In melanoma, tumor-reactive CD4 T cells have alsobeen associated with a good clinical outcome (Robbins P F et al., 2002),and more recently the same group showed that tumor specific CD4 T cellswere present in at least 20% of metastatic melanomas, and suggested thatthe infusion of TIL populations containing CD4 specific T cells couldenhance the efficacy of adoptive cell therapy (Friedman K M et al.,2012). In the same line of thought, it has been demonstrated in amelanoma patient that the adoptive cell transfer of CD4 T cells specificfor NYESO-1 antigen induces durable clinical remission and led toendogenous responses against non-targeted tumor antigens, suggesting thestimulation of immune responses by transferred CD4 T cells (Hunder N Net al., 2008).

In the field of peptide vaccination, it has been documented twenty yearsago, in a mouse model that the generation of a strong CD8 responseagainst a LCMV-derived peptide depended on the presence of CD4 helper Tcells (Fayolle C et al., 1991). These results have been more recentlyconfirmed in a clinical setting by the use of synthetic long peptides(SLP) in colorectal cancer, using P53 derived SLP (Speetjens F M et al.,2009), in vulvar intraepithelial neoplasia (Kenter G G et al., 2009) andcervical cancer patients (Welters M J et al., 2008) using HPV16-derivedSLP. In the case of vulvar neoplasia, clinical responses appeared to becorrelated with the induction of strong HPV16 specific immune responses.Synthetic long peptides containing immunogenic CD8 and CD4 tumorepitopes are therefore attractive tools to implement therapeutic cancervaccine.

One of the main issues in the field of long peptide vaccination in solidtumors is to identify immunogenic long peptides derived from relevanttumor associated antigens. Target antigens should be widely expressed,and able to induce robust CD8 and CD4 anti-tumor T cell responses. Inmelanoma, the Melan-A antigen fulfills these requirements and theinventors recently reported the efficiency of a Melan-A modified SLP, tocross-prime human tumor-reactive T cells (Chauvin J M et al., 2012).Another attractive target for melanoma vaccination would be the MELOE-1antigen (46 amino acids), specifically overexpressed in melanoma.Indeed, the inventors previously reported that the infusion of tumorinfiltrating lymphocytes (TIL) specific for the MELOE-1 antigen wasassociated with a prolonged relapse-free survival for HLA-A2 melanomapatients who received TIL therapy (Godet Y et al., 2008). Furthermore,they documented the presence of a large and tumor reactive CD8 T cellrepertoire in HLA-A2 melanoma patients (Godet Y et al., 2010) and thepresence of two class II epitopes in the vicinity of the class Iepitope, located at the C-terminal end of the polypeptide (Rogel A etal., 2011).

Despites these results, the identification of additional melanomaantigens with a documented immunogenic potential remains a major issueto address for melanoma immunotherapy.

SUMMARY OF THE INVENTION

In this study, the inventors find new epitopes located all along theMELOE-1 sequence and characterized by their T helper profile of CD4 Tcell response.

Thus, the invention relates to a melanoma antigen peptide comprising theamino acids sequence selected in the group consisting of SEQ ID NO:1,SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5 or afunction-conservative variant thereof. Moreover the invention alsorelates to a melanoma antigen peptide according to the invention for usein the prevention or the treatment of melanoma in patient.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Throughout the specification, several terms are employed and are definedin the following paragraphs.

As used herein, the term “peptide” refers to an amino acid sequencehaving less than 50 amino acids. As used herein, the term “peptide”encompasses amino acid sequences having less than 50 amino acids, lessthan 40 amino acids, less than 30 amino acids, less than 25 amino acids,less than 20 amino acids, less than 15 amino acids or less than 10 aminoacids.

Melanoma antigen peptides of the invention are described in table A.

TABLE A melanoma antigen peptides of the invention Nomenclatures used inSEQ ID number Sequences the patent application Peptide SEQ ID NO: 1SCVGYPDEATSREQFLPSEC MELOE-1₂₋₂₁ or 2-21 Peptide SEQ ID NO: 2VGYPDEATSREQFLPS MELOE-1₄₋₁₉ or 4-19 Peptide SEQ ID NO: 3 VGYPDEATSREQFLMELOE-1₄₋₁₇ or 4-17 Peptide SEQ ID NO: 4 GYPDEATSREQFLPSMELOE-1₅₋₁₉ or 5-19 Peptide SEQ ID NO: 5 PDEATSREQFLPSMELOE-1₇₋₁₉ or 7-19 Peptide SEQ ID NO: 6 PWHPSERISSTLNDECWPASLMELOE-1₂₆₋₄₆ or 26-46 Peptide SEQ ID NO: 7 RISSTLNDECWPASMELOE-1₃₂₋₄₅ or 32-45 Peptide SEQ ID NO: 8 RISSTLNDECWPAMELOE-1₃₂₋₄₄ or 32-44 Peptide SEQ ID NO: 9 ERISSTLNDECWPAMELOE-1₃₁₋₄₄ or 31-44 Peptide SEQ ID NO: 10 TSREQFLPSEGAACPPWHPSMELOE-1₁₁₋₃₀ or 11-30 Peptide SEQ ID NO: 11 REQFLPSEGAACPPWMELOE-1₁₃₋₂₇ or 13-27 Peptide SEQ ID NO: 12 EQFLPSEGAACPPWMELOE-1₁₄₋₂₇ or 14-27 Peptide SEQ ID NO: 13 QFLPSEGAACPPWMELOE-1₁₅₋₂₇ or 15-27 Peptide SEQ ID NO: 14 TSREQFLPSEGAAMELOE-1₁₁₋₂₃ or 11-23 Peptide SEQ ID NO: 15 SREQFLPSEGAACMELOE-1₁₂₋₂₄ or 12-24 Peptide SEQ ID NO: 16 PSEGAACPPWHPSERISSTLMELOE-1₁₈₋₃₇ or 18-37 Peptide SEQ ID NO: 17 AACPPWHPSERISSTLNDECWPASLMELOE-1₂₂₋₄₆ or 22-46 Peptide SEQ ID NO: 18 CPPWHPSERISSTLMELOE-1₂₄₋₃₇ or 24-37 Peptide SEQ ID NO: 19 CPPWHPSERISSTMELOE-1₂₄₋₃₆ or 24-36 Peptide SEQ ID NO: 20 MSCVGYPDEATSREQFLPSEGAACPPWMELOE-1₁₋₄₆ or 1-46 HPSERISSTLNDECWPASL Peptide SEQ ID NO: 21GHGHSYTTAEELAGIGILTVILGVL Melan-A_(16-40L)

As used herein, the term “antibody” refers to a protein capable ofspecifically binding an antigen, typically and preferably by binding anepitope or antigenic determinant or said antigen. The term “antibody”also includes recombinant proteins comprising the binding domains, aswell as variants and fragments of antibodies. Examples of fragments ofantibodies include Fv, Fab, Fab′, F(ab′)2, dsFv, scFv, sc(Fv)2,diabodies and multispecific antibodies formed from antibody fragments.

“Function-conservative variants” as used herein refer to those in whicha given amino acid residue in a protein or enzyme has been changed(inserted, deleted or substituted) without altering the overallconformation and function of the polypeptide. Such variants includeprotein having amino acid alterations such as deletions, insertionsand/or substitutions. A “deletion” refers to the absence of one or moreamino acids in the protein. An “insertion” refers to the addition of oneor more of amino acids in the protein. A “substitution” refers to thereplacement of one or more amino acids by another amino acid residue inthe protein. Typically, a given amino acid is replaced by an amino acidwith one having similar properties (such as, for example, polarity,hydrogen bonding potential, acidic, basic, hydrophobic, aromatic, andthe like). Amino acids other than those indicated as conserved maydiffer in a protein so that the percent protein or amino acid sequencesimilarity between any two proteins of similar function may vary and maybe, for example, from 70% to 99% as determined according to an alignmentscheme such as by the Cluster Method, wherein similarity is based on theMEGALIGN algorithm. A “function-conservative variant” also includes apolypeptide which has at least 60% amino acid identity as determined byBLAST or FASTA algorithms, preferably at least 75%, more preferably atleast 85%, still preferably at least 90%, and even more preferably atleast 95%, and which has the same or substantially similar properties orfunctions as the native or parent protein to which it is compared. Twoamino acid sequences are “substantially homologous” or “substantiallysimilar” when greater than 80%, preferably greater than 85%, preferablygreater than 90% of the amino acids are identical, or greater than about90%, preferably greater than 95%, are similar (functionally identical)over the whole length of the shorter sequence. Preferably, the similaror homologous sequences are identified by alignment using, for example,the GCG (Genetics Computer Group, Program Manual for the GCG Package,Version 7, Madison, Wisconsin) pileup program, or any of sequencecomparison algorithms such as BLAST, FASTA, etc.

The term “Major Histocompatibility Complex” (MHC) is a genericdesignation meant to encompass the histo-compatibility antigen systemsdescribed in different species including the human leucocyte antigens(HLA).

The term “melanoma” as used herein includes, but is not limited to, alltypes of melanocytes cancers at all stages of progression likemetastatic melanocyte cancer.

The term “treating” a disorder or a condition refers to reversing,alleviating or inhibiting the process of one or more symptoms of suchdisorder or condition. The term “preventing” a disorder or conditionrefers to preventing one or more symptoms of such disorder or condition.

As used herein, the term “patient” denotes a mammal, such as a rodent, afeline, a canine, and a primate. Preferably a patient according to theinvention is a human.

A “therapeutically effective amount” as used herein is intended for aminimal amount of active agent which is necessary to impart therapeuticbenefit to a patient. For example, a “therapeutically effective amountof the active agent” to a patient is an amount of the active agent thatinduces, ameliorates or causes an improvement in the pathologicalsymptoms, disease progression, or physical conditions associated withthe disease affecting the patient.

The term “adjuvant” as used herein refers to a compound or a mixturethat may be non-immunogenic when administered in the host alone, butthat augments the host's immune response to an antigen when administeredconjointly with that antigen.

Peptide, Fusion Protein and Uses Thereof

A first object of the invention relates to a melanoma antigen peptidecomprising the amino acids sequence selected in the group consisting ofSEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5 or afunction-conservative variant thereof.

In one embodiment, the melanoma antigen peptide has the sequence SEQ IDNO:5.

Melanoma antigen peptides of the invention, are generated from thepeptide MELOE-1 (SEQ ID NO:20) as described in the patent application WO2010 026165 and in table A.

In one embodiment, the melanoma antigen peptide of the invention is notthe peptide MELOE-1 (SEQ ID NO:20).

In one embodiment of the invention, by “antigen peptide” is meant apeptide capable of binding to HLA molecule and causing a cellular orhumoral response in a patient.

In a preferred embodiment of the invention, said antigen peptide maycomprise a specific motif such that the polypeptide binds an HLAmolecule and induces a CTL response.

In another preferred embodiment of the invention, said antigen peptidemay comprise a specific motif such that the polypeptide binds an HLAmolecule and induces a helper T cell response.

In one embodiment of the invention, said melanoma antigen peptides asdescribed here above are HLA-DQβ1*0201 restricted.

In one embodiment of the invention, said melanoma antigen peptides asdescribed here above are HLA-DQβ1*0202 restricted.

In one embodiment of the invention, said antigen peptide is an aminoacid sequence of less than 50 amino acids long that comprises the aminoacid motif SEQ ID NO: 1, 2, 3, 4 or 5 as defined here above.

In another embodiment of the invention, said antigen peptide is an aminoacid sequence of less than 45 amino acids long that comprises the aminoacid motif SEQ ID NO: 1, 2, 3, 4 or 5 as defined here above.

In another embodiment of the invention, said antigen peptide is an aminoacid sequence of less than 40 amino acids long that comprises the aminoacid motif SEQ ID NO: 1, 2, 3, 4 or 5 as defined here above.

In another embodiment of the invention, said antigen peptide is an aminoacid sequence of less than 30 amino acids long that comprises the aminoacid motif SEQ ID NO: 1, 2, 3, 4 or 5 as defined here above.

In another embodiment of the invention, said antigen peptide is an aminoacid sequence of less than 25 amino acids long that comprises the aminoacid motif SEQ ID NO: 1, 2, 3, 4 or 5 as defined here above.

In one embodiment, the antigen peptide according to the inventioncomprises at least 60% identity over said SEQ ID NO: 1, 2, 3, 4 or 5even more preferably at least 70%, at least 80%, at least 85%, at least90%, at least 95%, at least 97% and is still able to bind to HLAmolecule and causing a cellular or humoral response in a patient.

In another embodiment, the antigen peptide according to the inventionconsists in the amino acid sequence as set forth in SEQ ID NO: 1, 2, 3,4 or 5 or a variant thereof comprising at least 60%, preferably at least70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identitywith SEQ ID NO:1 and is still able to bind to HLA molecule and causing acellular or humoral response in a patient.

The invention also encompasses peptides that are function-conservativevariants of antigen peptides comprising SEQ ID NO: 1, 2, 3, 4 or 5 asdescribed here above.

Typically, the invention encompasses peptides substantially identical toantigen peptides comprising SEQ ID NO: 1, 2, 3, 4 or 5 in which one ormore residues have been conservatively substituted with a functionallysimilar residue and which displays the functional aspects of the antigenpeptides comprising SEQ ID NO: 1, 2, 3, 4 or 5 as described here above,i.e. being still able to bind to an HLA molecule in substantially thesame way as a peptide consisting of the given amino acid sequence.

Examples of conservative substitutions include the substitution of onenon-polar (hydrophobic) residue such as isoleucine, valine, leucine ormethionine for another, the substitution of one polar (hydrophilic)residue for another such as between arginine and lysine, betweenglutamine and asparagine, between glycine and serine, the substitutionof one basic residue such as lysine, arginine or histidine for another,or the substitution of one acidic residue, such as aspartic acid orglutamic acid or another.

The term “conservative substitution” also includes the use of achemically derivatized residue in place of a non-derivatized residue.“Chemical derivative” refers to a patient peptide having one or moreresidues chemically derivatized by reaction of a functional side group.Examples of such derivatized molecules include for example, thosemolecules in which free amino groups have been derivatized to form aminehydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups,t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Freecarboxyl groups may be derivatized to form salts, methyl and ethylesters or other types of esters or hydrazides. Free hydroxyl groups maybe derivatized to form O-acyl or O-alkyl derivatives. The imidazolenitrogen of histidine may be derivatized to form N-im-benzylhistidine.Chemical derivatives also include peptides which contain one or morenaturally-occurring amino acid derivatives of the twenty standard aminoacids. For examples: 4-hydroxyproline may be substituted for proline;5-hydroxylysine may be substituted for lysine; 3-methylhistidine may besubstituted for histidine; homoserine may be substituted for serine; andornithine may be substituted for lysine.

According to the invention, the antigen peptides of the invention can beobtained by synthesizing the peptides according to the method forpeptide synthesis known in the art.

In another embodiment, the antigen peptides of the invention may beincorporated into polytopes or fusion proteins. Two or more peptides ofthe invention can be joined together directly, or via the use offlanking sequences. Thompson et al., Proc. Natl. Acad. Sci. USA 92 (13):5845-5849 (1995), teaches the direct linkage of relevant epitopicsequences. The use of polytopes or fusion proteins as vaccines is wellknown. See, e.g. Gilbert et al., Nat. Biotechnol. 15 (12): 1280-1284(1997); Thomson et al., supra; Thomson et al., J. Immunol. 157 (2):822-826 (1996); Tam et al., J. Exp. Med. 171 (1): 299-306 (1990), all ofwhich are incorporated by reference. The Tam et al. reference inparticular shows that polytopes or fusion proteins, when used in a mousemodel, are useful in generating both antibody and protective immunity.

Thus, the invention also relates to a fusion protein comprising amelanoma antigen peptide according to the invention and a melanomaantigen peptide comprising the amino acids motif:

(SEQ ID NO: 22) - TX2NDECWPX9

wherein X2 is leucine, methionine, valine, isoleucine or glutamine andX9 is alanine, valine or leucine.

In one embodiment of the invention, said second melanoma antigen peptideis selected in the group consisting of peptides having the sequence SEQID NO: 23 to SEQ ID NO: 37 as described below.

Peptide SEQ ID NO 23 X₂ = L X₉ = A TLNDECWPA Peptide SEQ ID NO 24 X₂ = MX₉ = A TMNDECWPA Peptide SEQ ID NO 25 X₂ = V X₉ = A TVNDECWPAPeptide SEQ ID NO 26 X₂ = I X₉ = A TINDECWPA Peptide SEQ ID NO 27 X₂ = QX₉ = A TQNDECWPA Peptide SEQ ID NO 28 X₂ = L X₉ = V TLNDECWPVPeptide SEQ ID NO 29 X₂ = M X₉ = V TMNDECWPV Peptide SEQ ID NO 30 X₂ = VX₉ = V TVNDECWPV Peptide SEQ ID NO 31 X₂ = I X₉ = V TINDECWPVPeptide SEQ ID NO 32 X₂ = Q X₉ = V TQNDECWPV Peptide SEQ ID NO 33 X₂ = LX₉ = L TLNDECWPL Peptide SEQ ID NO 34 X₂ = M X₉ = L TMNDECWPLPeptide SEQ ID NO 35 X₂ = V X₉ = L TVNDECWPL Peptide SEQ ID NO 36 X₂ = IX₉ = L TINDECWPL Peptide SEQ ID NO 37 X₂ = Q X₉ = L TQNDECWPL

In another embodiment, the melanoma antigen peptide according to theinvention or the fusion protein according to the invention may be use inthe prevention or the treatment of melanoma in patient.

In one embodiment, said patient is genotyped with HLA-DQβ1*0201 orHLA-DQβ1*0202 alleles.

Nucleic Acids, Vectors, Recombinant Host Cells and Uses Thereof

Another object of the invention relates to a nucleic acid sequenceencoding a melanoma antigen peptide according to the invention or afusion protein according to the invention.

Another object of the invention relates to an expression vectorcomprising a nucleic acid sequence encoding a melanoma antigen peptideaccording to the invention or a fusion protein according to theinvention.

In one embodiment of the invention, said expression vector comprises thenucleic acid sequence corresponding to a melanoma antigen peptide havingthe sequence SEQ ID NO:1 to SEQ ID NO: 5.

According to the invention, expression vectors suitable for use in theinvention may comprise at least one expression control elementoperationally linked to the nucleic acid sequence. The expressioncontrol elements are inserted in the vector to control and regulate theexpression of the nucleic acid sequence. Examples of expression controlelements include, but are not limited to, lac system, operator andpromoter regions of phage lambda, yeast promoters and promoters derivedfrom polyoma, adenovirus, retrovirus, lentivirus or SV40. Additionalpreferred or required operational elements include, but are not limitedto, leader sequence, termination codons, polyadenylation signals and anyother sequences necessary or preferred for the appropriate transcriptionand subsequent translation of the nucleic acid sequence in the hostsystem. It will be understood by one skilled in the art that the correctcombination of required or preferred expression control elements willdepend on the host system chosen. It will further be understood that theexpression vector should contain additional elements necessary for thetransfer and subsequent replication of the expression vector containingthe nucleic acid sequence in the host system. Examples of such elementsinclude, but are not limited to, origins of replication and selectablemarkers. It will further be understood by one skilled in the art thatsuch vectors are easily constructed using conventional methods orcommercially available.

Another object of the invention is a host cell comprising an expressionvector as described here above.

According to the invention, examples of host cells that may be used areeukaryote cells, such as animal, plant, insect and yeast cells andprokaryotes cells, such as E. coli. The means by which the vectorcarrying the gene may be introduced into the cells include, but are notlimited to, microinjection, electroporation, transduction, ortransfection using DEAE-dextran, lipofection, calcium phosphate or otherprocedures known to one skilled in the art.

In a preferred embodiment, eukaryotic expression vectors that functionin eukaryotic cells are used. Examples of such vectors include, but arenot limited to, viral vectors such as retrovirus, adenovirus,adeno-associated virus, herpes virus, vaccinia virus, poxvirus,poliovirus; lentivirus, bacterial expression vectors, plasmids, such aspcDNA3 or the baculovirus transfer vectors. Preferred eukaryotic celllines include, but are not limited to, COS cells, CHO cells, HeLa cells,NIH/3T3 cells, 293 cells (ATCC# CRL1573), T2 cells, dendritic cells, ormonocytes.

In one embodiment, the nucleic acid sequence according to the inventionor the expression vector according to the invention or the host cellaccording to the invention may be use in the prevention or the treatmentof melanoma in patient.

Antibodies and Uses Thereof

Another object of the invention relates to an antibody or fragmentthereof that binds to the melanoma antigen peptide according toinvention.

In one embodiment of the invention, said antibody or fragment thereofbinds to melanoma antigen peptide having the sequence SEQ ID NO: 1 toSEQ ID NO: 5.

In one embodiment of the invention, said antibody is monoclonal. Inanother embodiment of the invention, said antibody is polyclonal.

Such antibodies may be easily prepared, for example, according to themethod described in “Antibodies: A laboratory manual”, Lane H. D. et al.eds, Cold Spring Harbor Laboratory Press, New York, 1989 or AntibodyEngineering: Methods and Protocols, 2003, Benny K. Lo.

MHC/Peptide Multimer

Another object of the invention relates to a MHC/peptide multimercomprising a melanoma antigen peptide as described here above. Accordingto the invention, said MHC/peptide multimer include, but are not limitedto, a MHC/peptide dimer, trimer, tetramer or pentamer.

In one embodiment of the invention, said MHC/peptide multimer is aHLA-class II/peptide multimer.

In another embodiment of the invention, said MHC/peptide multimer is aHLA-DQβ1*0201 II/melanoma antigen peptide multimer or aHLA-DQβ1*0202/melanoma antigen peptide multimer.

Methods for obtaining MHC/peptide tetramers are described in WO96/26962and WO01/18053, which are incorporated by reference.

In one embodiment of the invention, said MHC/peptide multimer can beused to visualise T cell populations that are specific for the complexHLA-DQβ1*0201/melanoma antigen peptide or HLA-DQβ1*0202/melanoma antigenpeptide multimer as described here above.

In another embodiment of the invention, said MHC/peptide multimer can beused for the detection and/or isolation by screening (in flow cytometryor by immunomagnetic screening) of T cell population that are specificfor a complex HLA/melanoma antigen peptide as described here above.

In another embodiment of the invention, said HLA-DQβ1*0201/melanomaantigen peptide or HLA-DQβ1*0202/melanoma antigen peptide multimer canbe used for the detection and/or isolation by screening (in flowcytometry or by immunomagnetic screening) of T cell population that arespecific for a complex HLA-DQβ1*0201/melanoma antigen peptide orHLA-DQβ1*0202/melanoma antigen peptide multimer as described here above.

Another object of the invention is beads coated with MHC/peptidemultimers as described here above.

Immunizing Composition and Uses Thereof

Another object of the invention relates to an immunising compositioncomprising:

(a) at least one melanoma antigen peptide as described here above or

(b) at least one fusion protein as described here above, or

(c) at least one nucleic acid sequence as described here above, or

(d) at least one expression vector as described here above, or

(e) at least one host cell as described here above, or

(f) at least one antibody as described here above.

In one embodiment, said immunising composition comprises a melanomaantigen peptide having a sequence SEQ ID NO: 1 to SEQ ID NO: 5.

The prophylactic administration of the immunizing composition of theinvention should serve to prevent or attenuate melanoma in a mammal In apreferred embodiment mammals, preferably human, at high risk formelanoma are prophylactically treated with the immunising composition ofthe invention. Examples of such mammals include, but are not limited to,humans with a family history of melanoma.

When provided therapeutically, the immunising composition of theinvention is provided to enhance the patient's own immune response tothe melanoma antigen present on the melanoma or metastatic melanoma.

In one embodiment of the invention, the peptides of the invention may beconjugated with lipoprotein or administered in liposomal form or withadjuvant.

In one embodiment, said immunising composition is a pharmaceuticalcomposition.

In such embodiment, said immunising composition, for human use,comprises at least one antigen peptide as described here above or atleast one antibody as described here above, together with one or morepharmaceutically acceptable carriers and, optionally, other therapeuticingredients. The carrier(s) must be “acceptable” in the sense of beingcompatible with the other ingredients of the composition and notdeleterious to the recipient thereof. The immunising compositions mayconveniently be presented in unit dosage form and may be prepared by anymethod well-known in the pharmaceutical art.

Immunising compositions suitable for intravenous, intradermal,intramuscular, subcutaneous, or intraperitoneal administrationconveniently comprise sterile aqueous solutions of the active agent withsolutions which are preferably isotonic with the blood of the recipient.Such compositions may be conveniently prepared by dissolving solidactive ingredient in water containing physiologically compatiblesubstances such as sodium chloride (e.g. 0.1-2.0M), glycine, and thelike, and having a buffered pH compatible with physiological conditionsto produce an aqueous solution, and rendering said solution sterile.These may be present in unit or multi-dose containers, for example,sealed ampoules or vials.

The immunising compositions of the invention may incorporate astabilizer. Illustrative stabilizers are polyethylene glycol, proteins,saccharides, amino acids, inorganic acids, and organic acids which maybe used either on their own or as admixtures. These stabilizers arepreferably incorporated in an amount of 0.11-10,000 parts by weight perpart by weight of active agent. If two or more stabilizers are to beused, their total amount is preferably within the range specified above.These stabilizers are used in aqueous solutions at the appropriateconcentration and pH. The specific osmotic pressure of such aqueoussolutions is generally in the range of 0.1-3.0 osmoles, preferably inthe range of 0.8-1.2. The pH of the aqueous solution is adjusted to bewithin the range of 5.0-9.0, preferably within the range of 6-8.

Additional pharmaceutical methods may be employed to control theduration of action. Controlled release preparations may be achievedthrough the use of polymer to complex or absorb the peptides of theinvention. The controlled delivery may be exercised by selectingappropriate macromolecules (for example polyester, polyamirio acids,polyvinyl, pyrrolidone, ethylenevinylacetate, methylcellulose,carboxymethylcellulose, or protamine sulfate) and the concentration ofmacromolecules as well as the methods of incorporation in order tocontrol release. Another possible method to control the duration ofaction by controlled-release preparations is to incorporate the antigenpeptides of the invention into particles of a polymeric material such aspolyesters, polyamino acids, hydrogels, poly(lactic acid) or ethylenevinylaceiate copolymers. Alternatively, instead of incorporating theseagents into polymeric particles, it is possible to entrap thesematerials in microcapsules prepared, for example, by coacervationtechniques or by interfacial polymerization, for example,hydroxy-methylcellulose or gelatin-microcapsules andpoly(methylmethacylate) microcapsules, respectively, or in colloidaldrug delivery systems, for example, liposomes, albumin microspheres,microemulsions, nanoparticles, and nanocapsules or in macroemulsions.

When oral preparations are desired, the compositions may be combinedwith typical carriers, such as lactose, sucrose, starch, talc magnesiumstearate, crystalline cellulose, methyl cellulose, carboxymethylcellulose, glycerin, sodium alginate or gum arabic among others.

Immunisation of a patient with the immunising composition of theinvention can be conducted by conventional methods, for example, in thepresence of conventional adjuvants. Examples of conventional adjuvantinclude, but are not limited to, metal salts, oil in water emulsions,Toll like receptors agonists, saponins, lipid A, alkyl glucosaminidephosphate, Freund's adjuvant, keyhole limpet haemocyanin (KLH), mannan,BCG, alum, cytokines such as IL-1, IL-2, macrophage colony stimulatingfactor, and tumor necrosis factor; and other substances that act asimmunostimulating agents such as muramyl peptides or bacterial cell wallcomponents, toxins, toxoids and TLR ligands.

The immunising composition can be administered by any route appropriatefor antibody production and/or T cell activation such as intravenous,intraperitoneal, intramuscular, subcutaneous, and the like. Theimmunising composition may be administered once or at periodic intervalsuntil a significant titre of anti-Nectin4 immune cells or anti-Nectin4antibody is produced. The presence of anti-Nectin4 immune cells may beassessed by measuring the frequency of precursor CTL (cytoxicT-lymphocytes) against the antigen peptides of the invention prior toand after immunization by specific tetramer labelling or by a CTLprecursor analysis assay. The antibody may be detected in the serumusing an immunoassay.

Antibodies directed to the antigens of the invention can also be useddirectly as anti-melanoma agents. To prepare antibodies, a host animalmay be immunized using melanoma antigen peptide as described here above.The host serum or plasma is collected following an appropriate time toprovide a composition comprising antibodies reactive to said antigenpeptides. The gamma globulin fraction or the IgG antibodies can beobtained, for example, by use of saturated ammonium sulfate or DEAFSephadex, or other techniques known to those skilled in the art. Theantibodies are substantially free of many of the adverse side effectswhich may be associated with other anti-cancer agents such aschemotherapy.

The immunising composition of the invention comprising antibodies asdescribed here above can be made even more compatible with the hostsystem by minimizing potential adverse immune system responses. This isaccomplished by removing all or a portion of the Fc portion of a foreignspecies antibody or using an antibody of the same species as the hostpatient, for example, the use of antibodies from human/human hybridomas.Humanized antibodies (i.e., nonimmunogenic in a human) may be produced,for example, by replacing an immunogenic portion of an antibody with acorresponding, but nonimmunogenic portion (i.e., chimeric antibodies).Such chimeric antibodies may contain the reactive or antigen bindingportion of an antibody from one species and the Fc portion of anantibody (nonimmunogenic) from a different species. Examples of chimericantibodies, include but are not limited to, nonhuman mammal-humanchimeras, rodent-human chimeras, murine-human and rat-human chimeras.

Methods for obtaining said antibodies, chimeric antibodies and humanizedchimeric antibodies are well-known in the art.

The immunising composition comprising the antibodies of the inventioncan also be used as a means of enhancing the immune response. Theantibodies can be administered in amounts similar to those used forother therapeutic administrations of antibody. For example, pooled gammaglobulin is administered at a range of about 1 mg to about 100 mg perpatient. Thus, antibodies reactive with the antigen peptides of theinvention can be passively administered alone or in conjunction withother anti-cancer therapies to a mammal afflicted with cancer. Examplesof anti-cancer therapies include, but are not limited to, chemotherapy,radiation therapy, adoptive immunotherapy therapy with TIL.

The antibodies or chimeric antibodies described herein may also becoupled to toxin molecules, radioisotopes and drugs by conventionalmethods. Examples of toxins to which the antibodies may be coupled toincluded, but are not limited to, ricin or diphtheria toxin. Examples ofdrugs or chemotherapeutic agents include, but are not limited to,cyclophosphamide or doxorubicin. Examples of radioisotopes, include, butare not limited to, 131I. Antibodies covalently conjugated to theaforementioned agents can be used in cancer immunotherapy for treatingmelanoma.

If the patient to be immunized is already afflicted with cancer ormetastatic cancer, the immunising composition of the invention can beadministered in conjunction with other therapeutic treatments. Examplesof other therapeutic treatments includes, but are not limited to,adoptive T cell immunotherapy, coadministration of cytokines or othertherapeutic drugs for cancer.

The dose of antigen peptide of the invention to be administered to apatient may be adjusted as appropriate depending on, for example, thedisease to be treated, the age and the body weight of said patient.Ranges of antigen peptides of the invention that may be administered areabout 0.001 to about 100 mg per patient, preferred doses are about 0.01to about 10 mg per patient.

The immunising composition of the invention may be evaluated first inanimal models, initially rodents, and in nonhuman primates and finallyin humans. The safety of the immunization procedures is determined bylooking for the effect of immunization on the general health of theimmunized animal (weight change, fever, appetite behavior etc.) andlooking for pathological changes on autopsies. After initial testing inanimals, cancer patients can be tested. Conventional methods would beused to evaluate the immune response of the patient to determine theefficiency of the immunising composition.

Another object of the invention relates to an immunising composition asdescribed above for use in the prevention or treatment of melanoma in apatient in need thereof

In another embodiment, said patient is genotyped with HLA-DQβ1*0201 orHLA-DQβ1*0202 alleles.

Antigen Presenting Cell

Another object of the invention is an antigen presenting cell comprisinga complex HLA antigen and a melanoma antigen peptide of the invention.

In one embodiment of the invention, said complex HLA antigen is aHLA-DQβ1*0201 or HLA-DQβ1*0202 antigen.

In one embodiment of the invention, said antigen presenting cell isderived from the patient to be treated.

The term “antigen presenting cell” (APCs) refers to any cell thatexpresses an HLA antigen capable of presenting the antigen peptide ofthe invention on its surface. Dendritic cells, which are reported tohave an especially high antigen-presenting ability, are preferred. Inanother embodiment, artificial APCs may also be used such mammaliancells (fibroblast, endothelial cells, keratinocytes), or cell lines.

In order to prepare such APCs of the invention, cells having anantigen-presenting ability are isolated from the patient to be treated,and pulsed ex vivo with at least one antigen peptide of the invention toform a complex with the HLA-DQβ1*0201 or HLA-DQβ1*0202 antigen.

In case dendritic cells are used, the APC of the invention can beprepared as follows. Lymphocytes are isolated from peripheral blood ofthe patient to be treated by Ficoll method; adherent cells are separatedfrom non-adherent cells; the adherent cells are then cultured in thepresence of GM-CSF and IL-4 to induce dendritic cells; and the dendriticcells are pulsed by culturing with at least one antigen peptide of theinvention to obtain the APCs of the invention. The dendritic cellsshould be exposed to the antigen peptide for sufficient time to allowthe antigens to be internalized and presented on the dendritic cellssurface. The resulting dendritic cells can then be re-administrated tothe patient to be treated. Such methods are described in WO93/208185 andEP0563485, which are incorporated by reference.

Another object of the invention is a composition for activeimmunotherapy comprising antigen presenting cells comprising a complexHLA antigen and a melanoma antigen peptide of the invention.

In one embodiment of the invention, said antigen presenting cellscomprise a complex HLA-DQβ1*0201 or HLA-DQβ1*0202 antigen and a melanomaantigen peptide of the invention.

Said APCs may be preferably contained in physiological saline, phosphatebuffered saline (PBS), culture medium, or the like. Administration maybe achieved, for example, intravenously, hypodermically, orintradermally.

Lymphocytes T and Uses Thereof

Another object of the invention relates to a T lymphocyte thatrecognizes specifically the melanoma antigen peptide of the invention ora fusion protein of the invention.

In one embodiment of the invention, said T lymphocyte is a T lymphocytehelper.

In another embodiment of the invention, said T lymphocyte isHLA-DQβ1*0201 or HLA-DQβ1*0202 restricted.

In another embodiment of the invention, said T lymphocyte is a T cellclone.

In another embodiment, said T lymphocyte is a genetically modified Tlymphocyte that expresses a TCR that recognizes specifically themelanoma antigen peptide of the invention.

Another object of the invention is a composition for adoptive therapycomprising said T lymphocytes as described here above that recognizesspecifically the melanoma antigen peptide of the invention or a fusionprotein of the invention.

In the case of melanoma, it has been observed that an adoptiveimmunotherapy wherein intratumoral T cell infiltrate taken from thepatient to be treated are cultured ex vivo in large quantities, and thenreturned into the patient achieves a therapeutic gain.

It is preferred that the T cells are contained in physiological saline,phosphate buffered saline (PBS), culture medium, or the like in order totheir stable maintain. Administration may be achieved, for example,intravenously or intra-tumoraly. By returning the T cells thatrecognizes specifically the antigen peptide of the invention into thepatient's body, the toxicity of said T cells, or the stimulation of CD8cytotoxic T cells by said cells towards tumor cells is enhanced in thepatient who is positive for the melanoma antigen peptide of theinvention. The tumor cells are destroyed and thereby the treatment oftumor is achieved.

Examples of where T-lymphocytes can be isolated, include but are notlimited to, peripheral blood cells lymphocytes (PBL), lymph nodes, ortumor infiltrating lymphocytes (TIL).

Such lymphocytes can be isolated from tumor or peripheral blood of theindividual to be treated by methods known in the art and cultured invitro. Lymphocytes are cultured in media such as RPMI or RPMI 1640 for2-5 weeks, preferably for 2-3 weeks. Viability is assessed by trypanblue dye exclusion assay. The lymphocytes are exposed to the antigenpeptide of the invention for all of the culture duration.

In a preferred embodiment the lymphocytes are exposed to the melanomaantigen peptide of the invention at a concentration of about 1 to about10 micrograms(μg)/ml for all the duration of lymphocyte culture.Cytokines may be added to the lymphocyte culture such as IL-2.

The melanoma antigen peptide of the invention may be added to theculture in presence of antigen presenting cells such as dendritic cellsor allogeneic irradiated cancer cell line cells.

After being sensitized to the peptide, the T-lymphocytes areadministered to the patient in need of such treatment.

Examples of how these sensitized T-cells can be administered to themammal include but are not limited to, intravenously, intraperitoneallyor intralesionally. Parameters that may be assessed to determine theefficacy of these sensitized T-lymphocytes include, but are not limitedto, production of immune cells in the patient being treated or tumorregression. Conventional methods are used to assess these parameters.Such treatment can be given in conjunction with cytokines or genemodified cells (Rosenberg, S. A. et al. (1992) Human Gene Therapy, 3:75-90; Rosenberg, S. A. et al. (1992) Human Gene Therapy, 3: 57-73).

Another object of the invention is a method for producing T lymphocytesthat recognize specifically a melanoma antigen peptide of the invention,said method comprising the steps of:

(a) stimulating peripheral blood mononuclear cells (PBMCs) or tumorinfiltrating lymphocytes (TIL) obtained from a patient with at least onemelanoma antigen peptide of the invention or a fusion protein of theinvention,

(b) enriching the population of T lymphocytes specific for the melanomaantigen peptide(s) used in (a),

(c) optionally cloning said population of T lymphocytes specific for themelanoma antigen peptide(s) used in (a).

Enrichment and/or cloning may be carried out by using an MHC/peptidemultimer as described here above. Cloning may also be carried out byconventional methods.

In one embodiment of the invention, the T lymphocytes that recognizespecifically a melanoma antigen peptide of the invention areHLA-DQβ1*0201 or HLA-DQβ1*0202 restricted. In such embodiment,enrichment and/or cloning may be carried out by using an HLA-DQβ1*0201or HLA-DQβ1*0202/peptide multimer as described here above.

Stimulation of PBMCs may be carried out with at least one melanomaantigen peptide of the invention alone, or presented by an antigenpresenting cell such as dendritic cells or allogeneic irradiated cancercell line cells. Typically, cytokines such as IL-2 may also be added tothe culture.

Another object of the invention is a composition for adoptive therapythat comprises lymphocytes that recognizes specifically the melanomaantigen peptide of the invention for preventing or treating melanoma ina patient in need thereof, wherein said T lymphocytes are to bere-administrated to the patient.

In one embodiment, said lymphocytes that recognizes specifically theantigen peptide of the invention are HLA-DQβ1*0201 or HLA-DQβ1*0202restricted.

The invention also relates to a method for preventing or treatingmelanoma in a patient in need thereof, comprising administering atherapeutically effective amount of

(a) at least one melanoma antigen peptide as described here above or

(b) at least one fusion protein as described here above, or

(c) at least one nucleic acid sequence as described here above, or

(d) at least one expression vector as described here above, or

(e) at least one host cell as described here above, or

(f) at least one antibody as described here above.

The invention also relates to a method for preventing or treatingmelanoma in a patient in need thereof, comprising administering atherapeutically effective amount of T lymphocytes that recognizesspecifically the melanoma antigen peptide of the invention. In oneembodiment, said T lymphocytes are HLA-DQβ1*0201 or HLA-DQβ1*0202restricted.

The invention also relates to a method for preventing or treatingmelanoma in a patient in need thereof, comprising administering atherapeutically effective amount of antigen presenting cells comprisinga complex HLA antigen and a melanoma antigen peptide of the invention.In one embodiment, said complex HLA/peptide is a complex HLA-DQβ1*0201or HLA-DQβ1*0202/antigen peptide of the invention.

The invention will be further illustrated by the following figures andexamples. However, these examples and figures should not be interpretedin any way as limiting the scope of the present invention.

FIGURES

FIG. 1: (A) Percentages of TNF-α producing CD4 T cells among positivemicrocultures. Fourteen days after PBMC stimulation with MELOE-1 wholepolypeptide (2-46), microcultures were re-stimulated with each indicatedpeptide during 5 hours. TNF-α production was then assessed by a doublelabeling TNF-α-CD4. Results were analyzed with a non-parametric test(Kruskal-Wallis) followed by a Dunns post-test. (B) Frequency ofmicrocultures containing CD4 T cells specific for MELOE-1 peptides(assessed by TNF-α intracellular staining after re-stimulation with the4 indicated peptides). 624 microcultures (from 7 healthy donors) wereanalyzed by a contingency table followed by a Fisher exact test.

FIG. 2: (A) Percentages of cytokine producing CD4 T cells among positivemicrocultures. Fourteen days after PBMC stimulation with MELOE-1 wholepolypeptide (2-46), microcultures were re-stimulated with each indicatedpeptide during 5 hours. IFN-γ (left panel) and IL4 (right panel)production was then assessed by a triple labeling IFN-γ-IL4-CD4. Resultswere analyzed with a non-parametric test (Kruskal-Wallis) followed by aDunns post-test. (B) Frequency of microcultures containing CD4 T cellsspecific for MELOE-1 peptides (assessed by IFN-γ or IL4 intracellularstaining after re-stimulation with the 3 indicated peptides). 576microcultures (from 10 melanoma patients) were analyzed by a contingencytable followed by a Fisher exact test.

FIG. 3: HLA-restricting element of MELOE-1 specific T cell clones andreactivity against HLA-matched melanoma cell lines. The HLA restrictionof MELOE-1 specific T cell clones was first assessed using anti-HLAblocking antibodies (upper panel). T cell clones were stimulated for 5hours in the presence of brefeldin A (10 μg/mL) either with peptidealone (10 μM) in an autopresentation assay and in presence or not ofblocking antibodies at a concentration of 12.5 μg/mL. HLA restrictionwas confirmed with HLA-matched B-EBV cell lines pulsed 2 hrs with thecognate peptide, at a ratio 1:2 (middle panel). Reactivity of each Tcell clone against HLA-class II expressing melanoma cells was assessed,in presence or not of exogeneous peptide (lower panel). After 5 hours ofstimulation, cells were then stained with APC-conjugated anti-CD4 mAb,fixed with 4% paraformaldehyde, labeled with PE-conjugated anti-TNF-αmAb and analyzed by flow cytometry.

FIG. 4: Class II epitopes are naturally processed from MELOE-1 wholeantigen. Autologous DC, were loaded (before or after fixation) withMELOE-1₂₋₄₆ (1 μM) or, as a negative control, with Melan-A_(16-40L)peptide (1 μM), and matured. T cell clones were then stimulated with DCat a ratio 1:1, during 5 h in presence of Brefeldin A, then stained withAPC-conjugated anti-CD3 mAb, fixed with 4% paraformaldehyde, labeledwith PE-conjugated anti-TNF-α mAb and analyzed by flow cytometry.Histograms illustrated the % of TNF-α producing cells among CD3 positiveT cells.

FIG. 5: Minimal peptides recognized by MELOE-1 specific CD4 T cellclones. MELOE-1 specific CD4 T cell clones were incubated with variousconcentrations of the indicated peptides during 5 hours in presence ofBrefeldin A. TNF-α production was assessed by intracellular labelingwith an anti-TNF-α specific antibody. The core peptide sequence isindicated in bold on each figure panel, and black circles illustrate thebest fitting peptide.

TABLE I MELOE-1 and MELOE-1 derived peptide sequences. Peptide SequencesSeq ID No MELOE-1 MSCVGYPDEATSREQFLPSEGAACPPWHPSERISSTLNDECWPASL 20MELOE-1₂₋₂₁  SCVGYPDEATSREQFLPSEG 1 MELOE-1₁₁₋₃₀          TSREQFLPSEGAACPPWHPS 10 MELOE-1₁₈₋₃₇                 PSEGAACPPWHPSERISSTL 16 MELOE-1₂₆₋₄₆                         PWHPSERISS

SL 6 MELOE-1₂₂₋₄₆                      AACPPWHPSERISS

SL 17

All the peptides were purchased from Millegen company (France), with apurity >85%. In bold are indicated the DR-11 (SEQ ID NO:18) and the DQ-6(SEQ ID NO:8) overlapping epitopes already described, and in italics isindicated the HLA-A2 restricted class I epitope (TLNDECWPA, SEQ IDNO:23)).

TABLE II Assessment of MELOE-1 CD4 T cell responses in PBMC from healthydonors. Microcultures containing MELOE-1 specific CD4+ T cells (TNF-αproduction) Donor MELOE-1₂₋₂₁ MELOE-1₁₁₋₃₀ MELOE-1₁₈₋₃₇ MELOE-1₂₆₋₄₆Class II HLA HD9 9/96 13/96 6/96 5/96 DPβ1*0902/1501 (1.4% ± 0.6) (2.4%± 1.5) (1.4% ± 0.6) (1.5% ± 0.5) DQβ1*0301 DRβ1*1104/1201 HD17 6/9626/96 7/96 10/96 DPβ1*0401/0301 (3.2% ± 1.9) (8.2% ± 6.5) (3% ± 0.7)(5.2% ± 4.1) DQβ1*0301/0603 DRβ1*1101/1301 HD22 1/96 2/96 0/96 2/96DPβ1*0401/1101 (1%) (3.9% ± 3.3) (1% ± 0.1) DQβ1*0202 DRβ1*0701 HD243/96 46/96 0/96 0/96 DPβ1*0401/0101 (1.3% ± 0.8) (2% ± 1.4)DQβ1*0501/0602 DRβ1*0101/1501 HD25 3/96 3/96 0/96 15/96 DPβ1*0401/0402(0.6% ± 0.1) (1.2% ± 0.7) (1.1% ± 0.9) DQβ1*0201 DRβ1*0301 HD27 0/965/96 0/96 0/96 DPβ1 NA (5% ± 3.4) DQβ1*501/0201 DRβ1*0101/0301 HD2830/48 14/48 0/48 0/96 DPβ1 NA (2.7% ± 1.2) (2.4% ± 0.-9) DQNA DRβ1*0301

PBMC from healthy donors were stimulated with 10 μM of MELOE-1. After 14days, the presence of CD4 T cells specific for the different regions ofMELOE-1 was assessed by restimulating cells with MELOE-1₂₋₂₁,MELOE-1₁₁₋₃₀, MELOE-1₁₈₋₃₇ and MELOE-1₂₆₋₄₆ peptides, followed byCD4/TNF-α double staining and flow cytometry analysis. Between bracketsis indicated the mean % of TNF-α producing CD4 T cells, in positivemicrocultures. NA: not available.

TABLE III Assessment of MELOE-1 CD4 T cell responses in PBMC frommelanoma patients. Microcultures containing MELOE-1 specific CD4+ Tcells Th1 responses (IFN-γ positive Th2 responses (IL4 positivemicrocultures) microcultures) MELOE- MELOE- MELOE- MELOE- MELOE- MELOE-1₂₋₂₁ 1₁₁₋₃₀ 1₂₂₋₄₆ 1₂₋₂₁ 1₁₁₋₃₀ 1₂₂₋₄₆ Pt ≠ 1 1/48 10/48  11/48  0/481/48 4/48 (0.7%) (1.4% ± 0.7) (4.8% ± 7.2) (1%) (0.8% ± 0.3) Pt ≠ 2 4/9616/96  2/96 0/96 0/96 0/96 (0.8% ± 0.2) (1.6% ± 1.2)  (0.6% ± 0.02) Pt ≠3 10/96 5/96 4/96 0/96 0/96 0/96 DPβ1*0201/ (1.7% ± 2.9) (16.2% ± 33.2)(1.9% ± 1.3) 2001 DQβ1*0303/ 0501 DRβ1*0101/ 0701 Pt ≠ 4 0/48 9/48 0/481/48 0/48 0/48 (0.9% ± 0.5) (0.7%) Pt ≠ 5 1/48 6/48 0/48 2/48 0/48 1/48(0.6%) (1.5% ± 1.1) (0.7% ± 0.1) (0.8%) Pt ≠ 6 0/48 5/48 0/48 0/48 0/480/48 (1.5% ± 1.9) Pt ≠ 7 0/48 0/48 28/48  0/48 0/48 7/48 (2.9% ± 3.5)(1.5% ± 0.7) Pt ≠ 8 0/48 2/48 1/48 0/48 0/48 1/48  (2.9% ± 0.04 ) (0.5%)(0.6%) Pt ≠ 9 0/48 0/48 0/48 1/48 9/48 3/48 (0.6%) (0.6% ± 0.09)  (0.7%± 0.15)

PBMC from melanoma patients were stimulated with 10 μM of MELOE-1. After14 days, the presence of CD4 T cells specific for the different regionsof MELOE-1 was assessed by restimulating cells with MELOE-1₂₋₂₁,MELOE-1₁₁₋₃₀ and MELOE-1₂₂₋₄₆ peptides, followed by CD4/IFN-γ doublestaining for the detection of Th1 responses, and by CD4/IL4 doublestaining for Th2 responses. Between brackets is indicated the mean % ofcytokine producing CD4 T cells, in positive microcultures.

TABLE IV TCR characterization and cytokine profile ofMELOE-1 specific CD4 T cell clones CDR3 beta  Cytokine  chain profileMELOE-1₂₋₂₁ specific CD4 T cell clone  (9C12-DQβ1*0202) V beta chainVβ2.1 TNF ^(high) CDR3 beta CSA SPDTHWGTDTQ YFG IFN ^(high) J beta chainJβ 2.3 IL2 ^(high) GM-CSF ^(high) IL4 ^(high) IL5 ^(low) IL13 ^(high)IL10 ^(neg) MELOE-1₁₁₋₃₀ specific CD4 T cell clone  (1A5-DRβ1*1101)V beta chain ND TNF ^(high) CDR3 beta ND IFN ^(high) J beta chain NDIL2 ^(high) GM-CSF ^(high) IL4 ^(low) IL5 ^(neg) IL13 ^(neg) IL10 ^(neg)MELOE-1₁₁₋₃₀ specific CD4 T cell clone  (5F9-DRβ1*0101) V beta chain NDTNF ^(high) CDR3 beta ND IFN ^(high) J beta chain ND IL2 ^(low)GM-CSF ^(high) IL4 ^(high) IL5 ^(neg) IL13 ^(high) IL10 ^(neg)MELOE-1₂₆₋₄₆ specific CD4 T cell clone  (4E2-DQβ1*0201) V beta chainVβ2.1 TNF ^(high) CDR3 beta CSA SGRRKFYEQ YFG IFN ^(high) J beta chainJβ 2.7 IL2 ^(high) GM-CSF ^(high) IL4 ^(high) IL5 ^(low) IL13 ^(high)IL10 ^(neg) CD4 T cell clones were stimulated for 5 hours in thepresence of brefeldin A (10 μg/mL) either with the cognate peptide (10μM) in an autopresentation assay. After 5 hours of stimulation, cellswere stained with APC-conjugated anti-CD4 mAb, fixed with 4%paraformaldehyde, labeled with PE- conjugated anti-cytokine mAb andanalyzed by flow cytometry.

EXAMPLE

Material & Methods

Cells

Blood samples from healthy subjects and melanoma patients wererespectively obtained from Etablissement Francais du Sang, Nantes,France and from the department of onco-dermatology, Nantes Hospital,France. Melanoma and B-EBV cell-lines were maintained in RPMI 1640(GIBCO) containing 10% fetal calf serum (FCS). Lymphocytes were grown inRPMI 1640 8% human serum (HS) with 50 or 75 IU/ml of recombinantinterleukin-2 (IL-2, Chiron, France) and 2 nM of L-Glutamin. Forexperiments using dendritic cells (DC), RPMI supplemented with 20 mg/mLof human albumin (LFB BIOMEDICAMENTS, France) was used to avoid peptidedegradation by serum proteases.

Reagents

Antibodies were purchased from BD Biosciences-France or from MiltenyiBiotec, France. Purified cytokines were purchased from CellGenix,Germany. The different peptides (Millegen, France, purity >85%) used inthis study are described in Table I. HLA-A*0201/MELOE-1₃₆₋₄₄ monomerswere generated by the recombinant protein facility of our institute (SFR26).

Dendritic Cells Generation and Loading Monocytes were purified from PBMCof healthy donors by a CD14-enrichment kit, according therecommendations of the supplier (Stem Cell, France). Immature dendriticcells (iDC) were generated by culturing monocytes in RPMI supplementedwith 20 mg/mL of human albumin, 1000 IU/mL of GM-CSF and 200 IU/mL ofIL-4 for 5 days. Then, iDC were pulsed with the whole MELOE-1 (1 μM)protein or the modified Melan-A₁₆₋₄₀ A27L as negative control (1 μM) andmatured with 20 ng/mL of TNF-α and 50 μg/mL of PolyI:C for 4 hours at37° C. Finally they were fixed for 1 minute with PBS/0.015%glutaraldehyde. Alternatively, iDC were first matured, fixed and thenpulsed with antigens at the same concentration.

Stimulation of MELOE-1 Specific T Cells

PBMC from healthy donors or melanoma patients (2·10⁵ cells/well) werecultured for 14 days with 10 μM of MELOE-1 whole antigen (46 aminoacids) in RPMI medium supplemented with 8% HS, 50 IU/ml of rIL-2(Chiron, France) and L-Glutamin, in 96-well multiplates. Micro cultureswere then restimulated individually with each overlapping peptide(MELOE-1₂₋₂₁, MELOE-1₁₁₋₃₀, MELOE-1₁₈₋₃₇, MELOE-1₂₆₋₄₆ or MELOE-1₂₂₋₄₆for melanoma patients) in the presence of 10 μg/mL brefeldin A for 5hours and the percentage of CD4⁺ specific T cells was assessed by TNF-α,IFN-γ or IL-4 intracellular staining A negative control without peptidewas included in all experiments.

Alternatively, MELOE-1 specific CD4+ T cell clones were stimulated byautologous MELOE-1 loaded and matured DC at a 1:1 ratio.

T Cell Cloning and TCR Characterization

Polyclonal cultures containing specific CD4+ T cells were cloned bylimiting dilution as previously described (Gervois N. et al., 2000).After 2 weeks, each clone was checked for peptide specificity by TNFproduction assay. For TCR sequencing, RNA from 5·10⁶ T cell clones wasextracted with RNable reagent (Eurobio, France) according to thesupplier's instructions. Reverse transcriptions, PCR amplifications andsequencing were performed as described (Davodeau F. et al, 2001). Weused the TCR nomenclature established by Arden et al. (Arden et al.,1995).

TNF Production Assay

CD4⁺ T cell clones were cultured for 5 hours at 37° C. in the presenceof the recognized 20-mer peptide. Culture supernatants were harvestedand TNF was measured in a biological assay using cytotoxicity againstWEHI 164 clone 13 (Espevik T. et al., 1986).

Cytokine Intracellular Staining

Lymphocytes were stimulated for 5 hours in the presence of brefeldin A(10 μg/mL) either with peptide alone (10 μM) in an autopresentationassay or with B-EBV or HLA-class II expressing melanoma cells pulsed 2hours with the cognate peptide, at a ratio 1:2. In some experiments,blocking mAb against HLA-DP (clone B7.21 from Dr Charron, UMR940,Paris), HLA-DQ (clone SPVL3, Beckman Coulter) or HLA-DR (clone L243, BDBiosciences) were added at a concentration of 12.5 μg/ml. Cells werethen stained with APC-conjugated anti-CD4 mAb, fixed with 4%paraformaldehyde, labeled with PE-conjugated anti-cytokine mAb andanalyzed by flow cytometry.

Statistical Analyses

Statistical analyses were done with GraphPad Prism® software. Bar graphswere used to compare frequencies of T cells specific for MELOE-1-derivedpeptides, in all donors and patients and were analyzed by a contingencytable followed by a Fisher exact test. Scatter-dot graphs were made tocompare the percentage of TNFα positive cells among positivemicrocultures and were analyzed with a non-parametric test(Kruskal-Wallis followed by a Dunns post-test).

Results

Frequency and Distribution of MELOE-1 Specific CD4 responses in healthydonor's PBMC Stimulated with MELOE-1 antigen

Our purpose was to look for the existence of class II helper epitopesall along MELOE-1 sequence (SEQ ID NO: 20), in order to document theimmunogenicity of the different regions of this melanoma antigen. Westimulated 2·10⁷ PBMC from seven healthy donors with MELOE-1 wholeantigen and tested, after a 14-day culture period the presence of CD4 Tcells specific for each region of the protein. Microcultures werescreened for TNFα production by CD4+ T cells, after restimulation withfour MELOE-1 derived overlapping peptides (Table I), in anautopresentation assay. As shown in table II, all donors exhibited CD4responses against at least 1 out of 4 overlapping peptides. Responsesagainst the N-terminal region of MELOE-1 (2-21) were detected in 6/7donors, with rather low frequencies (from 1 to 9% of positivemicrocultures containing between 0.6 to 5.6% of TNFα producing CD4 Tcells), unless in HD28 healthy donor, who exhibited 62% of positivemicrocultures. The region 11-30 appears especially immunogenic, with CD4specific responses detected in each tested donor (from 2 to 48% ofpositive microcultures containing between 0.7 to 24% of TNFα producingCD4 T cells), and with very high frequencies in three donors (HD17, HD24and HD28). On the contrary, the central region 18-37, containing analready described DR11-restricted epitope (24-37) located just at theend of this 20-mer peptide (Rogel et al., 2011), induced specificresponses in microcultures deriving from only 2/7 donors (HD9 and HD17,both expressing the DR11 element). In these two donors, we detected 6and 7% of positive microcultures, containing between 0.6 to 3.7% of TNFαproducing CD4 T cells. Finally, the C-terminal region (26-46),containing an already described DQ6-restricted epitope (Rogel et al.,2011), was recognized by stimulated microcultures from 4 out of 7 donors(all do not expressing the DQ6 element), with frequencies ranging from 2to 16% of microcultures containing between 0.5 and 16% of TNFα producingCD4 T cells. Overall, the frequency of MELOE-1₁₁₋₃₀ positivemicrocultures was significantly higher than frequency of microculturesspecific for the three other regions of MELOE-1 (FIG. 1B) (p<0.0001).The two terminal regions (2-21 and 26-46) were equivalent in terms offrequencies of positive microcultures, and these two regions inducedsignificantly more responses than the central region 18-37 (FIG. 1A).Nonetheless, the mean fractions of CD4 reactive T cells induced inpositive microcultures were not significantly different from a region toanother (FIG. 1B).

Frequency, Distribution and Th Profile of MELOE-1 Specific CD4 Responsesin Melanoma Patient's PBMC Stimulated with MELOE-1 Antigen

In order to confirm the immunogenicity of each MELOE-1 regions inmelanoma patients, we stimulated melanoma patients PBMC with the MELOE-1whole protein, and tested the reactivity of stimulated lymphocytestowards the three most immunogenic regions: 2-21, 11-30, and 22-46. Forthis study, instead of challenging microcultures with the 18-37 peptide,that appeared poorly immunogenic, we extended the C-terminus region from26-46 to 22-46, in order to also detect responses to our previouslydescribed HLA-DR11-restricted epitope (24-37). Indeed, the location ofthis epitope just at the end of the 18-37 peptide could be deleteriousfor the detection of specific responses in additional DR contexts, andwe previously showed that CD4 T cells specific for MELOE-1₂₄₋₃₆ epitopewere efficiently induced by 22-46 peptide stimulation (Rogel et al.,2011). We tested the induction of CD4 specific responses from MELOE-1stimulated PBMC of 10 melanoma patients. We documented CD4 responsesspecific for the central region of MELOE-1 (11-30) for 7/9 patients,whereas responses specific for MELOE-1₂₋₂₁ and MELOE-1₂₂₋₄₆ wererespectively detected in 4/9 and 5/9 patients (Table III). Theseresponses were mainly Th1 responses (IFN-g production) while lessfrequent Th2 responses specific for the three regions of MELOE-1 weredetected in 3/9 patients for MELOE-1₂₋₂₁, 2/9 patients for MELOE-1₁₁₋₃₀and 5/9 patients for MELOE-1₂₂₋₄₆ (Table III). Considering the variousregions of MELOE-1, Th1 responses specific for the N-term region ofMELOE-1 (2-21) were significantly less frequent than those specific forthe central region (p<0.0001) and the C-term region (p=0.0001), withrespectively 3.3%, 10.8% and 9.6% of 576 tested microculutures (FIG.2A). Concerning Th2 responses, much less frequent, the C-term regionappeared to induce more frequently the growth of IL-4 producing CD4 Tcells than the two other regions (FIG. 2A). As observed for healthydonors, even if the frequencies were different, the mean fraction ofreactive T cells (Th1 and Th2) induced in positive microcultures werenot significantly different from a region to another (FIG. 2B). Insummary, stimulation of patient's PBMC with MELOE-1 induced Th1responses specific for diverse epitopes located all along the proteinsequence, and among the different regions, the central region (11-30)and the C-term region (22-46) appeared to be especially immunogenic interm of frequency of responses.

Production and Characterization of CD4 T Cell Clones Specific for theDifferent Regions of MELOE-1

In order to formally characterize the recognized epitopes, we derivedCD4 T cell clones specific for each region of MELOE-1 by limitingdilution, from microcultures of healthy donors or melanoma patients,containing at least 0.5% of specific CD4 T cells. We succeeded to deriveCD4 specific T cell clones from HD17, HD22, HD25 and Pt≠3 microcultures,which were reactive against MELOE-1₂₋₂₁ (HD22), MELOE-1₁₁₋₃₀ (HD17 andPt≠3) and MELOE-1₂₆₋₄₆ (HD25). From each cloning experiment, we obtainedbetween one and ten reactive CD4 T cell clones, that turned out to bethe same clonotype after CDR3B sequencing (Table IV). A single CD4 Tcell clone for each specificity was used for further experiments. TheHLA-restriction was determined for each T cell clone, first by usingHLA-class II blocking monoclonal antibodies (FIG. 3, upper panel), andfurther by testing the recognition of HLA-matched B-EBV cell linesloaded with each recognized long peptide (FIG. 3 middle panel). The twoT cell clones named 9C12 and 4E2, derived from HD22 and HD25 andspecific for MELOE-1₂₋₂₁ and MELOE-1₂₆₋₄₆ were restricted by theHLA-DQβ1*0202 and the DQβ1*0201 molecules respectively. As, these twodonors were homozygous for the HLA-DQ locus, a single HLA-DQ matchedB-EBV cell line was tested to confirm the HLA restriction of these twoCD4 T cell clones. As shown on FIG. 3 (upper panel), the two other Tcell clones 1A5 and 5F9 recognized the 11-30 region of MELOE-1, in aHLA-DR context. The use of HLA-matched B-EBV cell lines allowed toprecise that 1A5 T cell clone was restricted by the HLA-DRβ1*1101molecule and the 5F9 T cell clone by the HLA-DRβ1*0101 molecule.

We further tested the reactivity of these CD4 T cell clones againstHLA-matched melanoma cell lines positive for meloe expression, by qPCRanalysis. All the melanoma cell lines tested expressed HLA-DQ and DR atthe cell surface. All the T cell clones were reactive againstHLA-matched melanoma cell lines when loaded with the cognate peptide(FIG. 3, lower panel, black bars). The two DQ2-restricted T cell cloneswere reactive against peptide-loaded DQβ1*0201 and 0202 melanoma celllines. In absence of peptide, only the 4E2 DQβ1*0201-restricted T cellclone was able to recognize unloaded M77 melanoma cell line (alsoDQβ1*0201), but not the DQβ1*0202 melanoma cell line, M88. Similarly,the DR1-restricted T cell clone 5F9 also recognized one of the DRβ1*0101melanoma cell line, in the absence of exogenous peptide (M101).

We also documented the T helper profile of each T cell clone, bystimulating the CD4 T cell clones with the cognate peptide, andanalyzing cytokine production. All the clones expressed Th1 cytokines(TNFα, IFNγ, IL2 and GM-CSF). On the contrary, these clones differ intheir expression of Th2 cytokines. Indeed, the two DQ2 restricted T cellclones (9C12 and 4E2) and the DR1 restricted one (5F9) also stronglyexpress two Th2 cytokines (IL4, and IL13), whereas the DR11-restricted Tcell clone only weakly expressed IL4 (Table IV). None of the CD4 T cellexpresses IL10 or IL5 at a significant level.

Processing of the Recognized Epitopes from Autologous DC Loaded withMELOE-1 Antigen

Initial PBMC stimulation was carried out with MELOE-1 whole antigen, andthus we assume that CD4 T cell responses were generated against peptidesnaturally processed by monocytes. Nonetheless, we could not formallyexclude that the 14-day culture period artificially generated shorterclass II epitopes that elicited CD4 T cell responses. Thus, it remainedcrucial to assess that all these new epitopes were naturally processedby autologous dendritic cells loaded with MELOE-1 whole protein, inserum-free medium. To this end, we loaded autologous iDC with 1 μM ofMELOE-1 antigen in serum-free medium, in presence of maturating agents,and fixed these DC before stimulation of the CD4 specific T cell clones.In these conditions, the four T cell clones were reactive againstMELOE-1 loaded autologous DC (FIG. 4, left panel), whereas we could notdetect any reactivity of CD4 T cell clones to DC loaded with anirrelevant long peptide, synthesized in the same conditions(Melan-A_(16-40L)).

As an additional control, we loaded autologous DC with MELOE-1 after DCfixation, and we could observe only a weak recognition by specific CD4 Tcell clones, indicating that only a small fraction of the protein hadbeen externally degraded into shorter peptides (FIG. 4, right panel).Thus, the four new epitopes identified by PBMC stimulation, arenaturally processed from MELOE-1 antigen.

Characterization of the Minimal Recognized Epitopes

Our T cell clones were reactive against 20-mer peptides that areprobably not the exact peptides naturally processed. In order toformally identify the minimal recognized epitopes, we tested shorterpeptides derived from each of the MELOE-1 recognized regions, chosen onthe basis of the core peptide sequence supposed to be recognized by theT cell clones (indicated in bold on FIG. 5).

Three shorter peptides were better recognized by the DQ2-restricted 9C12T cell clone, the shortest one being MELOE-1₇₋₁₉ (13-mer), recognizedwith an EC50 of 100 nM. The deletion of the two amino acids in C-termstrongly reduces T cell clone recognition. The other DQ2-restricted Tcell clone (4E2) better recognized a 14-mer peptide (31-44) also with anEC50 of 100 nM, and also recognized to a lower extent the 32-44 epitope(FIG. 5) previously described in the HLA-DQβ1*0603 context (Rogel etal., 2011). Concerning the DR-restricted T cell clones, optimal shorterpeptides were 13-mer peptides, MELOE-1₁₅₋₂₇ for the DRβ1*1101 restrictedT cell clone 1A5 (EC50=100 nM), and MELOE-1₁₁₋₂₃ or MELOE-1₁₂₋₂₄ for theDRβ1*0101 restricted one (FIG. 5). Nonetheless all of these clonesrecognized a series of shortened peptides, thus we cannot formallyassess that the shortest ones will be the exact peptides naturallypresented on class II molecules.

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The invention claimed is:
 1. A melanoma antigen peptide having less than40 amino acids comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQID NO:5; or a fusion peptide comprising said amino acid sequence and amelanoma antigen peptide comprising the amino acids motif:TX2NDECWPX9(SEQ ID NO: 23), wherein X2 is leucine, methionine, valine,isoleucine or glutamine and X9 is alanine, valine or leucine; or acomposition comprising said melanoma antigen peptide or said fusionpeptide.
 2. A method of treating melanoma in a patient in need thereof,comprising administering to the patient a therapeutically effectiveamount of an agent selected from the group consisting of: i) a melanomaantigen peptide according to claim 1, ii) a fusion protein according toclaim 1, or iii) a composition according to claim
 1. 3. The methodaccording to claim 2 wherein the patient is genotyped with HLA-DQβ1*0201or HLA-DQβ1*0202 alleles.